Advances in the clinical management of individuals infected with the human immunodeficiency virus type 1 (HIV-1) have been able to reduce viral titers below the detection limits of some early-generation HIV-1 assays. More specifically, highly active anti-retroviral drug therapy (HAART) can reduce the viral load down to a level approaching 50 HIV-1 RNA copies/ml, a level substantially below the 400-500 copies/ml threshold of some previous detection assays. This fact, together with a desire to monitor and maintain low viral titers, necessitated the development of improved quantitative assays for measuring HIV-1 RNA. (Elbeik et al., J. Clin. Micro. 38:1113-1120 (2000)) Complicating matters, however, is the fact that useful quantitative assays must be capable of accurately measuring a range of genetically diverse HIV-1 variants.
Three classes of HIV-1 have developed across the globe: M (major), O (outlying) and N (new). Among the M group, which accounts for greater than 90% of reported HIV/AIDS cases, viral envelopes have diversified so greatly that this group has been subclassified into nine major clades including A-D, F-H, J and K, as well as several circulating recombinant forms. Subtypes within the HIV-1 O group are not clearly defined, and the diversity of sequences within the O group is nearly as great as the diversity of sequences in the HIV-1 M group. Phylogenetic analyses of the gag and env genes have failed to reveal clades of O group viruses as clearly as the clades detected in the M group. Subtypes and sub-subtypes of the HIV-1 M group are thought to have diverged in humans following a single chimpanzee-to-human transmission event. In contrast, the HIV-1 O and N groups are each thought to have resulted from separate chimpanzee-to-human transmission events. Of the completely sequenced HIV-1 genomes, nearly 20% have a mosaic structure consisting of at least two subtypes, yet another potential complication for ultrasensitive HIV-1 detection assays. (Spica et al., J. Antimicrobial Chemotherapy 51:229 (2003).)
Most viral load monitoring is currently performed in the developed Western World where the Glade B (i.e., “subtype B” hereafter), which represents only about 3% of HIV infections worldwide, predominates. Importantly, the HIV-1 viral subtypes are expanding in different geographical regions, thereby imposing an additional requirement for broad detection capacity on detection and viral load monitoring assays. Accordingly, there is a need for ultrasensitive HIV-1 detection assays which are capable of accurately measuring the full range of HIV-1 subtypes. The present invention addresses this need.
An example quantitative HIV-1 assay, performed using real-time monitoring of a nucleic acid amplification reaction, has been described in published International Patent Application WO 2003106714.